User:Delna M. John/Sandbox1 Glucosamine 6 Phosphate Synthase



Abstract

C. albicans is a fungus normally present on the skin and within the mucous membrane. Despite its common presence, any stimuli for overgrowth can induce the invasion of C. albicans into the throat, intestines, and heart valves as it travels down the bloodstream. The fungus can be present in various morphologies such as rounded buds (yeast), pseudohyphae, and hyphae (mycelia). However, it is the hyphal form that induces the invasion into tissue. Glucosamine 6 phosphate (GlcNAc) synthase is an enzyme that catalyzes the first step in the pathway that ultimately results in hyphal formation. As a result, UDP-GlcNAc, a precursor of chitin, is generated. However, the specific mechanisms for the production of UDP-GlcNAc by this enzyme are not clearly understood. Eukaryotic GlcNAc synthase is a dimer of two dimers and displays a tetrameric structure. Each subunit is composed of two domains, GAH and ISOM. GAH is involved in glutamine hydrolysis while the ISOM domain is directly involved with the isomerization of fructose 6 phosphate to glucose 6 phosphate. We have analyzed and constructed a 3D physical model of GlcNAc synthase that focuses on the ISOM domain (346-712 aa) in complex with UDP-GlcNAc and fructose 6 phosphate using the computer software RasMol. The secondary structure of each subunit involves beta sheets (light pink) with hydrogen bonds (white) that provide stabilization to the model. In addition, we have selected residues involved in the tetramerization of this enzyme, an intermolecular interaction that is not observed in prokaryotes. Our physical model depicts residues involved in tetramerization (524-527). Other contacts related to tetramerization between the subunits are present in residues 391-445 (yellow). We have also selected fructose 6 phosphate (light green) in complex with the enzyme. Residues of the enzyme that are in close proximity to this molecule; Glu591, Lys588, His607 (light blue) reveal a possible binding region. Since the experimental procedures involved the replacement of GlcN-6P with Fructose-6P, and the combination of the obtained crystal structures with and without Fructose-6P using computer software, we only see the presence of one Fructose-6P ligand binding despite four possible binding sites. Finally, UDP-GlcNAc (magenta) molecule and surrounding amino acids 474-492 (royal blue) are represented to highlight the binding pocket. Although UDP-GlcNAc is present in complex only on chains A and B, the binding pocket is evident on all four chains. This is due to the fact that there were two crystal structures obtained for the synthase. Crystals were grown both in the absence of UDP-GlcNAc and in the presence of UDP-GlcNAc (Konariev et all 2007). Both crystal structures were used to manually form the final model within our referred PDB structure (2PUT) using computer software, and thus we see the UDP-GlcNAc binding in one chain, while the other dimer chain of each pair is free of the ligand. Although the distinct tetramerization, fructose interaction sites, and UDP-GlcNAc binding pocket selected in the physical model represent important areas in the development of the invasive mycelia form of C. albicans, there are numerous other factors such as phosphorylation sites present on the GAH domain, amino acids involved in amido transfer, and other regions which are also highly significant. Further investigation could reveal possible competitive inhibition for the binding region of fructose and tetramerization sites which can the synthase and thus prevent the formation of UDP-GlcNAc and ultimately inhibiting mycelia transition. As we further explore the structure of the synthase and other molecules in the pathway, we can further elucidate such mechanisms.

C. Albicans as a Pathogen – Yeast to Mycelia

Candidiasisis a mycosis (fungal infection) that encompasses minor rashes to life threatening situations, especially in Immunocompromisedpatients. It is most commonly caused by the opportunistic pathogen Candida Albicans, a fungus commonly found on our skin and mucous membrane. The transition of the fungus to the pathogenic form is induced by environmental conditions and secondary messengers that initiate a corresponding transition from the yeast to mycelial (hyphae) form. It is the mycelial form is associated directly with invasion into tissue. In order to make the metamorphosis into mycelia, it is essential to activate the molecular pathways that lead to the formation of chitin, as a higher chitin content is necessary in the cell wall of hyphae. The first pathway that is initiated results in the synthesis of UDP-GlcNAc, a precursor of Chitin.

GlcN6P-Synthase in the mycelial transformation

The metabolic pathway involves four steps, each catalyzed by a different enzyme. Glucose-amine-6-Phosphate synthase catalyzes the first step of the pathway resulting in the conversion of Fructose-6-Phosphate to Glucosamine-6-Phosphate The conversion itself involves two steps; glutamine hydrolysis and the isomerisation of Fructose-6-Phosphate. Both steps are correlated directly with the two separate domains of the synthase respectivelythe N-terminus GAH and C -terminus ISOM. The exact mechanism of the synthase activity is not fully understood, but there are quite a few areas of significance to the actual mechanism of catalysis by the synthase. The interaction between the two domains is related to a hydrophobic channel that ensures sufficient ammonia transfer between the two, and there are  various sites, especially those related to phosphorylation (an inducer of greater chitin content) within the GAH domain. Although the action of the enzyme involves the dynamic use of the entire structure, it is the ISOM domain that participates directly in the binding of ligands such as GlcN-6P, Fructose-6P, and the negative feedback inhibitor UDP-GlcNAc. We have examined the structure and functional mechanisms of the ISOM domain in detail and deduced some of the vital sites of ligand interaction and interaction within the protein structure itself that might pose value in the full knowledge and understanding of its metabolic pathway.